1H ENDOR Spectra Research Articles

Distribution of H^upbeta Hyperfine Couplings in a Tyrosyl Radical Revealed by 263 GHz ENDOR Spectroscopy

^1H ENDOR spectra of tyrosyl radicals (Y^bullet) have been the subject of numerous EPR spectroscopic studies due to their importance in biology. Nevertheless, assignment of all internal ^1H hyperfine couplings has been challenging because of substantial spectral overlap. Recently, using 263 GHz ENDOR in conjunction with statistical analysis, we could identify the signature of the H^{upbeta _2} coupling in the essential Y_{122} radical of Escherichia coli ribonucleotide reductase, and modeled it with a distribution of radical conformations. Here, we demonstrate that this analysis can be extended to the full-width ^1H ENDOR spectra that contain the larger H^{upbeta _1} coupling. The H^{upbeta _2} and H^{upbeta _1} couplings are related to each other through the ring dihedral and report on the amino acid conformation. The 263 GHz ENDOR data, acquired in batches instead of averaging, and data processing by a new “drift model” allow reconstructing the ENDOR spectra with statistically meaningful confidence intervals and separating them from baseline distortions. Spectral simulations using a distribution of ring dihedral angles confirm the presence of a conformational distribution, consistent with the previous analysis of the H^{upbeta _2} coupling. The analysis was corroborated by 94 GHz ^2H ENDOR of deuterated Y_{122}^bullet. These studies provide a starting point to investigate low populated states of tyrosyl radicals in greater detail.

Probing the ternary complexes of indoleamine and tryptophan 2,3-dioxygenases by cryoreduction EPR and ENDOR spectroscopy.

We have applied cryoreduction/EPR/ENDOR techniques to characterize the active-site structure of the ferrous-oxy complexes of human (hIDO) and Shewanella oneidensis (sIDO) indoleamine 2,3-dioxygenases, Xanthomonas campestris (XcTDO) tryptophan 2,3-dioxygenase, and the H55S variant of XcTDO in the absence and in the presence of the substrate l-Trp and a substrate analogue, l-Me-Trp. The results reveal the presence of multiple conformations of the binary ferrous-oxy species of the IDOs. In more populated conformers, most likely a water molecule is within hydrogen-bonding distance of the bound ligand, which favors protonation of a cryogenerated ferric peroxy species at 77 K. In contrast to the binary complexes, cryoreduction of all of the studied ternary [enzyme-O2-Trp] dioxygenase complexes generates a ferric peroxy heme species with very similar EPR and 1H ENDOR spectra in which protonation of the basic peroxy ligand does not occur at 77 K. Parallel studies with l-Me-Trp, in which the proton of the indole nitrogen is replaced with a methyl group, eliminate the possibility that the indole NH group of the substrate acts as a hydrogen bond donor to the bound O2, and we suggest instead that the ammonium group of the substrate hydrogen-bonds to the dioxygen ligand. The present data show that substrate binding, primarily through this H-bond, causes the bound dioxygen to adopt a new conformation, which presumably is oriented for insertion of O2 into the C2−C3 double bond of the substrate. This substrate interaction further helps control the reactivity of the heme-bound dioxygen by “shielding” it from water.

ENDOR Spectroscopy Shows that QA Remains in the Same Orientation Upon Reduction in Reaction Centers from Rhodobacter Sphaeroides

It has been, recently, proposed that QA moves and rotates ∼60° upon its reduction.1 Here we investigated possible changes using ENDOR spectroscopy that provides a very sensitive (to ∼0.01 A) probe for the binding site of QA•-.2 Identical RC samples were made - (a) one frozen in the dark (ground state) and then illuminated generating D•+QA•- and (b) one frozen under illumination (excited state) which trapped D•+QA•- in ∼70% of the RCs at 80 K. Figure 1 shows the resultant 1H ENDOR spectra of QA•-. The peaks labeled L1, L2 and L3 correspond to the two H-bonds to QA•-.2,3 Essentially no differences in the ENDOR spectra were observed indicating that the interactions of QA•- with the protein are the same in the ground state as in the excited state. These results are irreconcilable with the proposed rotation.1 Thus, QA is preset in an environment that favors its reduction.1 Heinent et al. (2007), J. Am. Chem. Soc. 129, 15935. 2 Flores et al. (2007), Biophys. J. 92, 671. 3 Sinnecker et al. (2006), Phys. Chem. Chem. Phys. 8, 5659.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Intermediates Trapped during Nitrogenase Reduction of N⋮N, CH3−NNH, and H2N−NH2

A high population intermediate has been trapped on the nitrogenase active site FeMo cofactor during reduction of N2. In addition, intermediates have been trapped during reduction of CH3-N=NH by the alpha-195Gln variant and during reduction of H2N-NH2 by the alpha-70Ala/alpha-195Gln variant. Each of these trapped states shows an EPR signal arising from an S = 1/2 state of the FeMo cofactor. 15N ENDOR shows that each intermediate has a nitrogenous species bound to the FeMo cofactor, with a single type of N seen for each bound intermediate. The g tensors are unique to each intermediate, g(e) = [2.084, 1.993, 1.969], g(m) = [2.083, 2.021, 1.993], g(l) = [2.082, 2.015, 1.987], as are the 15N hyperfine couplings at g1, which suggests that three distinct stages of NN reduction may have been trapped. The 1H ENDOR spectra show that the N2 intermediate is at a distinct and earlier stage of reduction from the other two, so at least two stages of NN reduction have been trapped. Some possible structures of the hydrazine intermediate are presented.

W-Band ENDOR Investigation of the Manganese-Binding Site of Concanavalin A: Determination of Proton Hyperfine Couplings and Their Signs

W-band (95 GHz) pulsed EPR and electron−nuclear double resonance spectroscopic techniques were used to investigate the manganese S1 binding site of the protein concanavalin A in a frozen solution and in a single crystal. 1H ENDOR spectra were collected by using the Davies ENDOR sequence, whereas Mims ENDOR was applied to record 2H ENDOR spectra. Different EPR transitions were selected by performing the ENDOR measurements at different magnetic fields within the EPR spectrum. This selection, combined with the large thermal polarization achieved at magnetic fields of ∼3.4 T at low temperatures, allowed the assignment of ENDOR signals to their respective MS manifolds, thus providing the sign of the hyperfine couplings. The exchangeable and nonexchangeable protons were differentiated by comparing 1H and 2H ENDOR spectra of a solution of the protein prepared in D2O and of crystals soaked in D2O. The two imidazole protons, located on the carbons flanking the Mn-bound nitrogen, are magnetically equivalent, situated 3.56 Å from the Mn2+ and their hyperfine coupling is purely dipolar. The four protons of the two water ligands are all inequivalent, and four values of A∥ and A⊥ were determined. All possible combinations of these values yield distances in the range of 2.67 to 3.24 Å and aiso between −1.13 and 1.37 MHz. By limiting aiso to positive values, only four distances remain, 2.67, 2.76, 2.99, and 3.24 Å, corresponding to the four Mn−Hwater distances.

On the electronic structure of [Pt(4,4′-X2-bipy)Cl2]0/−/2−: an electrochemical and spectroscopic (UV/Vis, EPR, ENDOR) study

A series of complexes of general formula [Pt(4,4′-X2-bipy)Cl2] (bipy = 2,2′-bipyridine; X = NH2, OEt, Me, H, Ph, Cl or CO2Me) has been prepared and their redox chemistry and UV/Vis spectroscopy examined. The half-wave potential of the first reduction process from cyclic voltammetry varies linearly with the Hammett parameter of X, and also with the MLCT maximum from UV/Vis spectroscopy. The first reduction processes of the complexes with X = OEt, Me, H, Ph, Cl or CO2Me are reversible and these complexes undergo a second quasi-reversible or irreversible reduction at potentials 580–760 mV more negative. The reduced, 17 e– species are characterised by EPR spectroscopy and the total platinum contribution (5d and 6p) to the SOMOs is calculated to be only ca. 7–12%. The bulk of the unpaired electron density is carried in the bipy ligand π* system and orientation-selective 1H ENDOR spectra of [Pt(4,4′-X2-bipy)Cl2]– (X = H or CO2Me) show that C5 and C5′ carry the greatest spin density among the four ring C(H) positions. The second reduction product of [Pt(4,4′-(CO2Me)2-bipy)Cl2] is EPR silent indicating spin-pairing of the two reduction electrons in the same orbital in this particular complex.

ESR Study of High Spin States Derived from Tribenzoylenebenzene

By reduction of tribenzoylenebenzene 4 with lithium in 2-methyltetra-hydrofuran (MTHF) an ion pair and a cluster with S = 1 were obtained whereas with potassium or sodium in MTHF in the presence of dicyclohexano-18-crown-6 (or a solvent of strong solvating power such as diglyme or hexamethyl-phosphoric triamide), different species with S ⩾ 1/2 were detected. The ESR spectra of the randomly oriented pluri-anions in glassy matrix showed the existence of triplet species assigned to a dianion [42(.−)(M+)2] and aggregates [4.−(M+)n4.−] and quartet species [42(−)(M+)34.−]. The quartet species was attributed to intermolecular high-spin clusters with the relatively small zero-field splitting parameter D and vanishing E value. Further reduction led to the formation of a more persistent species with a quartet ground state characterized by the zero-field parameters D = + 0.0215 cm−1 and E = 0 cm−1, assigned to a triradical-trianion [43(.−)(M+)3], generated by three-electron doping per a single tribenzoylenebenzene molecule. The observed 1H-ENDOR spectra and INDO calculations also confirmed the above identification. 43(.−) is a stable pluri-anionic high-spin molecule in solution.

Making hyperfine selection in Mims ENDOR independent of deadtime

A form of remote echo-detected ENDOR can be performed by adding an additional π pulse to the detection phase of a Mims three-pulse, stimulated-echo ENDOR experiment. The resulting four-pulse experiment, denoted refocused Mims (ReMims) ENDOR, retains the hyperfine selectivity of the Mims technique, but makes it possible to obtain arbitrarily short preparation intervals, τ 1, independent of the spectrometer deadtime. As a result, without resonator redesign, it is possible to study a wider range of hyperfine values without distortions from “blind-spots”. The hyperfine selectivity of the ReMims ENDOR sequence is demonstrated on the 1H and 14N ENDOR spectra from a single crystal of Cu(glycine) 2. Use of the ReMims protocol to augment the basic Mims and Davies ENDOR sequences is shown in two different types of situation: the sign of a 9 MHz coupled proton is assigned by the use of the implicit-TRIPLE effect in the proton ENDOR spectrum of nitrile hydratase; the true lineshape of the proton ENDOR spectrum of fluorometmyoglobin is determined without distortions by blindspots.

EPR and ENDOR studies on monoanionic complexes of 1,2-semiquinones with 52Cr, 53Cr(III)

Monoanionic complexes of chromium(III) like [Cr(sq) 2(cat)] −, where the ligands are anionic radicals such as 1,2-semiquinones and catecholates unsubstituted and 3,5-di- tert-butyl-substituted were detected after interaction of chromate anion (Cr(VI), natural isotope, I = 0 or 53Cr-enriched isotope, I = 3 2 with 1,2-catechol or 3,5-di- tert-butyl-1,2-catechol in methanolic, ethanolic, or aqueous solution under both anaerobic and aerobic conditions. The very low 〈g〉 values ( g = 1.972) obtained in methanol for both complexes, and the very strong metal hyperfine coupling, A 53 Cr = 25.71 G and A 53 Cr = 25.17 G for the complex with the ligands 1,2-catechol eand 3,5-di- tert-butyl-1,2-catechol, respectively, suggest a ground state localized on the metal ion. The unpaired electron could be localized in a molecular orbital with a large metal character. The isotropic spectra obtained at room temperature as well as at low temperature suggest an essential octahedral arrangement around the chromium ion. The 1H ENDOR spectra confirm the octahedral geometry of the complexes.

EPR and ENDOR investigation of the primary electron acceptor radical anion QA.- in iron-depleted photosystem II membrane fragments.

Photosystem II (PS II) membrane fragments were treated with trypsin at pH = 7.4 followed by incubation with o-phenanthroline and lithium perchlorate. This procedure removes and/or decouples the non-heme Fe2+ associated with the quinones QA and QB in the PS II reaction center (RC). Treatment of such samples (referred to as iron-depleted) with sodium dithionite or illumination in the presence of dichlorophenol indophenol (DCIP) and sodium ascorbate yielded EPR spectra similar to those of the plastoquinone-9 (PQ-9) radical anion generated in organic solvents. Q-band EPR yielded the principal values of the g-tensor for PQ-9.- in 2-propanol and QA.- in PS II. Electron nuclear double resonance (ENDOR) experiments were performed both on PQ-9.- in vitro and on QA.- in the iron-depleted PS II samples. For the former a complete set of isotropic 1H hyperfine coupling constants and hyperfine tensors of the two methyl groups and the alpha-proton were obtained. On the basis of H/D exchange experiments two different hydrogen bonds could be detected in frozen solution that are formed between the carbonyl oxygens of the radical and protons from the surrounding solvent molecules. The hydrogen bond distances were estimated using the point-dipole model. 1H-ENDOR spectra of QA.- in iron-depleted PS II samples have been measured in buffers made in H2O and D2O. The spectrum in deuterated buffer allowed the determination of two different methyl group hyperfine tensors. Differences detected between the spectra in protonated and deuterated buffer reveal the hyperfine tensors of two exchangeable protons belonging to hydrogen bonds between the oxygens of QA and specific protein residues. The assignment of these hydrogen bonds in PS II is discussed and compared with the situation found in the bacterial reaction center.

31P and 1H powder ENDOR and molecular orbital study of a CO3 3? ion in X-irradiated carbonate containing hydroxyapatites

An X-irradiated synthetic carbonate-containing apatite powder is examined with EPR and ENDOR. At low microwave powers, the room-temperature EPR spectrum contains a major contribution of a signal with g values: gx= 2.0045, gy= 2.0034 and gz= 2.0014. In a related 13C-enriched sample, the radical was shown to exhibit a hyperfine interaction with one carbon nucleus. The 13C hyperfine tensor values are: Ax= 263 MHz, Ay= 263 MHz and Az= 423MHz. The radical is assigned to a CO33– molecular ion. It is demonstrated by means of CNDO/II and INDO calculations that by lowering the symmetry of the CO33– ion from C3v to Cs, an orthorhombic g tensor can be obtained. However, the deviation from axial symmetry for the 13C hyperfine tensor is so small that it is not measurable on a powder specimen. The thus-calculated spin-Hamiltonian parameters are in very good qualitative and quantitative agreement with the experimental ones, adding strong evidence for the assignment of the observed signal to a CO33– radical.At low temperatures, both 31P and 1H ENDOR spectra are recorded for different settings of the magnetic field (i.e. when the magnetic field is swept through the EPR CO33– spectrum). By a careful analysis of the ENDOR powder spectra using computer simulations based on the ‘orientation-selection’ principle, a detailed model for the CO33– ion could be proposed. In this way, it is established unambiguously that the CO33– ion substitutes for a phosphate group in the hydroxyapatite lattice, with a vacancy on the nearest hydroxy-group site. In addition, some deductions can be made about the substitution mechanism according to which the precursor of the CO33– radical (i.e. a carbonate ion) is incorporated into the apatitic lattice.

EPR and ENDOR detection of compound I from Micrococcus lysodeikticus catalase.

We present the first EPR and ENDOR examination of a catalase compound I (Cat I), the one formed by peracetic acid treatment of Micrococcus lysodeikticus catalase. The Cat I rapid-passage EPR signal (g perpendicular eff = 3.32; g parallel eff approximately 2) appears quite different from those reported previously for the compounds I from horseradish peroxidase (HRP I) and chloroperoxidase. Nonetheless, all three signals can be explained by the same model for exchange coupling between an S = 1 oxoferryl [Fe = O]2+ moiety and a porphyrin pi-cation radical (S' = 1/2) (Schulz, C. E., et al. (1979) FEBS Lett. 103, 102-105). The signal for Cat I is unlike those for the two peroxidases in that it reflects a ferromagnetic rather than antiferromagnetic exchange. Preliminary 1H ENDOR spectra for Cat I appear to differ from the proton (1H) ENDOR spectra of HRP I; the latter, along with the 14N ENDOR spectra, indicate that the porphyrin radical in HRP I exhibits a predominantly A2u-like state having large spin densities on porphyrin N and C(beta). The proton ENDOR spectrum of Cat I is insensitive to H/D exchange, which indicates that the [Fe = O]2+ moiety is not protonated. Consideration of the EPR results for a series of compounds I suggests that the sign and magnitude of the exchange parameter (J) is correlated with the nature of the proximal axial ligand.

ENDOR Spectroscopic Study of Macroradicals in Irradiated Oriented Nylon-6

Abstract Macroradicals formed by hydrogen atom elimination in uniaxially and biaxially oriented nylon-6 have been investigated at low temperature using a Q-band ENDOR technique. Hyperfine coupling constants were obtained from the observed 1H ENDOR spectra for the protons located at α-, β-, and γ-positions relative to the unpaired electron in the radical -CO-NH-CH-CH2-CH2-. Principal values for α-proton anisotropic coupling tensor have been determined. A degree of angular disorientation of the radical centers -CH- along the axes of biaxially oriented polymer was evaluated. The protons of the β-methylene group were found to be magnetically nonequivalent and therefore the plane of the free carbon was twisted. The deviation from the ideally planar transconfiguration corresponds to the rotation around the Cα-Cβ bond on average angle 11°. A change in β-proton ENDOR spectrum has been revealed under mechanical loading of oriented specimens. The results obtained evidenced for be appearance of reversible torsional ...

ENDOR and TRIPLE Studies of Diphenyl Nitroxide (DPNO)

Abstract ENDOR and triple resonance (GTR) of diphenyl nitroxide (DPNO) were observed in solution; it was demonstrated that DPNO is an interesting model radical forming a three-spin system (electron and 1H- and 14N-nuclear spins). The detected 14N-ENDOR belongs to one of the smallest hyperfine cases (AN=0.9653 mT). Its optimum temperature was 210 K, higher than that of the 1H, which is due to the large anisotropic hyperfine interaction of 14N. In the 1H-ENDOR spectra the effect of cross relaxation was observed, as in the case of the 14N-ENDOR lines; their temperature variations of intensity showed two maximum regions, one due to the optimum energy relaxation in the 14N-spin system and the other due to the energy relaxation inherent in the 1H-spin system. In the triple studies new phenomena were pointed out: An enhancement saturation effect due to the large hyperfine interaction and an intensified enhancement or de-enhancement effect due to the cross relaxation.

Single-crystal EPR and ENDOR study of nitrogenase from clostridium pasteurianum

The molybdenum-iron (MoFe) protein of nitrogenase from Clostridium pasteurianum forms monoclinic crystals in space group P2 1. The unit cell comprises two molecules, each of which possesses a molecular twofold symmetry axis and binds two chemically identical molybdenum-iron cofactors (M centers). Thus, there are four magnetically distinct M centers within the unit cell related by two different symmetry operations: a molecular twofold axis of symmetry in the bc ∗ plane relates the two EPR centers within a molecule; a crystallographic twofold screw axis of symmetry along the b axis of the unit cell relates the two different molecules in a unit cell. Single-crystal EPR studies at X-band (9.5 GHz) and Q-band (35 GHz) microwave frequencies have been employed to determine g tensors in the crystallographic frame for the four magnetically distinct M centers. Determination of these tensors has been achieved by a novel procedure that relies heavily on the symmetry relations between sites; it rests primarily on measurements that only involve rotation of the magnetic field parallel to a single plane of the crystal and uses g values from frozen-solution samples instead of additional rotations. Single-crystal 1H ENDOR spectra of the M centers are presented.

An ENDOR Study of Nitrosyl Myoglobin Single Crystals

Abstract14N, 15N and 1H ENDOR spectra were taken from single crystals of 15NO ligated myoglobin at about 5 K in order to probe the spatial arrangement of the protons and nitrogens from the environment interacting with the Fe‐N‐O complex and to analyze its bonding configuration. The 14N hyperfine (20.4 MHz, 15.2 MHz and 16.8 MHz) and quadrupolar tensors (+1.67 MHz, −0.86 MHz, −0.98 MHz) of NE2 of the proximal histidine expand the previously reported picture from ESR analysis showing a distorted NO bond configuration. 15N data of the N(NO) ligand indicate, however, an orientation of the NO ligand differing from previous ESR reports. The single crystal spectra are applied to an interpretation of the more complex powder‐type spectra. Nine different 1H interaction tensors have been analyzed and assigned on the basis of theoretical simulation of the single crystal gamut of line positions. For the distal NE2 bound histidine proton, a small position distribution, combined with a strong anisotropy of interaction, is shown to broaden the ENDOR patterns beyond detection. The consequences of this finding with respect to the tetrameric molecule hemoglobin are discussed.

1H and 14N ENDOR spectra of tert-butylphenylnitroxide

1H and 14N ENDOR spectra of tert-butylphenylnitroxide were observed in ethylbenzene solution. The experimental conditions for detecting optimum 1H and 14N ENDOR lines were quite different. The 1H hyperfine coupling constants and their signs are discussed from the viewpoints of molecular orbital calculations, general triple resonance, CRISP (cross relaxation intensity sequence pattern) method, and steric hindrance. In view of the large anisotropic hyperfine interaction between the nitrogen and the unpaired electron, detection of 14N ENDOR in nitroxide radicals is rather difficult, so that a TM110 mode cavity was utilized in this experiment. The observed 14N hyperfine coupling constant 11.86 G at −80 °C is one of the smallest values observed to date for nitroxide radicals.

Electron spin resonance and ENDOR reinvestigation of iminoxyl radicals from 1-halogenofluorenone oximes

In a reinvestigation of 1-halogenofluorenone iminoxyl radicals, 1H and 14N ENDOR spectra have been obtained from the chloro-compound. The e.s.r. spectra of the series of radicals (X = H, F, Cl and Br) have been analysed in detail by means of computer-fitting procedures. Contrary to previous interpretations, the spectra of the chloro-compound must be attributed to a single species, namely the anti form. Magnitude and sign of the coupling constant of the proton in position 8 are in accordance with through-space spin-density transfer. The effects of halogen substitution on the ENDOR response are discussed.

ChemInform Abstract: ENDOR Study of a One‐Electron 2:18 Reduced Fluoropolytungstate.

AbstractThe 19F and 1H ENDOR spectra of the one‐electron reduced (H2W18F6O56)9‐ polyanion show that the unpaired electron is trapped inside the equatorial rings of the 2:18 structure.

EPR and endor studies of cobaloxime(II)

EPR and ENDOR studies were carried out on bis (dimethylglyoximato) cobalt (II) in various frozen solvents. The anisotropic EPR spectra, from which the principal values of the g and 59Co hyperfine tensors were determined, enabled orientationally selected 1H ENDOR spectra to be obtained. In a tetrahydrofuran (THF)/toluene matrix, THF was shown to be axially coordinated to the metal ( R Co-O(THF) = 0.22 nm) with the THF plane at an angle of ≈55° to the yz plane and retention of symmetry about the xz plane. 1H ENDOR of the dimethylglyoximate ligands yielded information about the H-bond geometry and in-plane assignment of the principal values of the g tensor.